Qian X, Kulig E, Jin L, Lloyd R V
Department of Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.
Endocrinology. 1998 Apr;139(4):2058-67. doi: 10.1210/endo.139.4.5955.
The D-type cyclins (D1, D2, and D3) are involved in progression through the G1 phase of the cell cycle and are induced as part of the delayed early response to growth factor stimulation. To better understand the role of D-type cyclins in pituitary cell function and the regulatory role of growth factors in the cell cycle, we analyzed the expression and regulation of D-type cyclins in normal and neoplastic rat pituitary cells. Immunocytochemical and RT-PCR analyses showed expression of all three D-type cyclins in the normal pituitary, with higher percentages of positive cells by immunocytochemistry in the nuclei of normal pituitaries (D1, 20-30%; D2, 50-60%; D3, 70-80%), compared with GH3 cells. In the normal pituitary, there were significantly higher levels of cyclins D2 and D3 in PRL, GH, LH, and TSH cells, compared with ACTH cells. Cyclin D1 protein was not detected in GH3 cells, while D2 was present in less than 1 percent and D3 in 10-15 percent of GH3 cells. There were low levels of cyclin D1 and D2 messenger RNA expression in GH3 cells, by RT-PCR. When dissociated rat pituitary cells were cultured in the presence of basic fibroblast growth factor (5.6 nM) for 3 days, cyclin D2 was up-regulated 2-fold in normal PRL cells (control, 33 +/- 1%; treated, 68 +/- 2%). Similarly, bFGF treatment stimulated cyclin D3 expression 3-fold in GH3 cells (control, 15 +/- 1%; treated, 44 +/- 1%). Treatment of GH3 cells with 5-aza-2'-deoxycytidine, which induces gene demethylation, produced marked increases in cyclin D2 and D3 expression. Transfection of mouse cyclin D1 complementary DNA, driven by a cytomegalovirus promoter into GH3 cells, led to ectopic cyclin D1 expression; and there was a slight stimulation of cell proliferation and increased apoptosis in GH3 cells. These results indicate that there is a differential expression of various D-type cyclins in different types of normal pituitary cells and between normal pituitary and GH3 cells. Growth factors, such as bFGF and demethylation increased D-type cyclin expression, whereas ectopic overexpression of cyclin D1 stimulates cell proliferation and increases apoptosis in GH3 pituitary tumor cells.
D型细胞周期蛋白（D1、D2和D3）参与细胞周期G1期的进程，并作为对生长因子刺激的延迟早期反应的一部分被诱导产生。为了更好地理解D型细胞周期蛋白在垂体细胞功能中的作用以及生长因子在细胞周期中的调节作用，我们分析了正常和肿瘤性大鼠垂体细胞中D型细胞周期蛋白的表达和调节情况。免疫细胞化学和逆转录-聚合酶链反应（RT-PCR）分析显示，正常垂体中所有三种D型细胞周期蛋白均有表达，与GH3细胞相比，正常垂体细胞核中免疫细胞化学阳性细胞百分比更高（D1，20%-30%；D2，50%-60%；D3，70%-80%）。在正常垂体中，与促肾上腺皮质激素（ACTH）细胞相比，催乳素（PRL）、生长激素（GH）、促黄体生成素（LH）和促甲状腺激素（TSH）细胞中细胞周期蛋白D2和D3的水平显著更高。在GH3细胞中未检测到细胞周期蛋白D1蛋白，而D2存在于不到1%的GH3细胞中，D3存在于10%-15%的GH3细胞中。通过RT-PCR检测，GH3细胞中细胞周期蛋白D1和D2信使核糖核酸（mRNA）表达水平较低。当解离的大鼠垂体细胞在碱性成纤维细胞生长因子（bFGF，5.6 nM）存在下培养3天时，正常PRL细胞中细胞周期蛋白D2上调2倍（对照，33±1%；处理后，68±2%）。同样，bFGF处理使GH3细胞中细胞周期蛋白D3表达增加3倍（对照，15±1%；处理后，44±1%）。用诱导基因去甲基化的5-氮杂-2'-脱氧胞苷处理GH3细胞，可使细胞周期蛋白D2和D3表达显著增加。将由巨细胞病毒启动子驱动的小鼠细胞周期蛋白D1互补DNA转染到GH3细胞中，导致细胞周期蛋白D1异位表达；并且GH3细胞的细胞增殖略有刺激，凋亡增加。这些结果表明，不同类型的正常垂体细胞之间以及正常垂体与GH3细胞之间，各种D型细胞周期蛋白存在差异表达。生长因子，如bFGF和去甲基化增加了D型细胞周期蛋白的表达，而细胞周期蛋白D1的异位过表达刺激了GH3垂体肿瘤细胞的细胞增殖并增加了凋亡。
